Association Between Kinetics of Early Biofilm Formation and Clonal Lineage in Escherichia coli

BackgroundEscherichia coli biofilm formation has mostly been assessed in specific pathogenic E. coli groups. Here, we assessed the early biofilm formation (EBF), i.e., adhesion stage, using the BioFilm Ring Test® on 394 E. coli clinical isolates (EC) [196 consecutively isolated (CEC) in 2016 and 198 ESBL-producing E. coli (ESBLEC) isolated in 2015]. Then, biofilm-forming ability was contrasted with phylogroups, clonotypes (fumC-fimH), and sequence types (STs), all being used to define clones, virulence factors (VF), and FimB.ResultAccording to both biofilm production levels at 2, 3, and 5 h, and EBF kinetics over 5 h, CEC and ESBLEC isolates segregated into three EBF groups: strong (G1), moderate (G2), and weak (G3) producers. At 2 h, strong producers were more frequent among CEC (n = 28; 14.3%) than among ESBLEC (n = 8; 4%) (P = 0.0004). As CEC and ESBLEC isolates showed similar individual EBF kinetics in each group, a comparison of isolate features between each group was applied to gathered CEC and ESBLEC isolates after 2 h of incubation, 2 h being the most representative time point of the CEC and ESBLEC isolate segregation into the three groups. Phylogroup B2 displayed by 51.3% of the 394 isolates was more frequent in G1 (77.8%) than in G3 (47.6%) (P = 0.0006). The 394 isolates displayed 153 clones, of which 31 included at least three isolates. B2-CH14-2-ST127, B2-CH40-22-ST131, B2-CH52-5/14-ST141, and E-CH100-96-ST362 clones were associated with G1 (P < 0.03) and accounted for 41.7% of G1 isolates. B2-CH40-30-ST131 clone was associated with G3 (P < 0.0001) and accounted for 25.5% of G3 isolates. VF mean was higher among G1 than among G3 isolates (P < 0.001). FimB-P2 variant was associated with G1 (P = 0.0011) and FimB-P1 variant was associated with G3 (P = 0.0023). Clone, some VF, and FimB were associated with EBF, with clonal lineage being able to explain 72% of the variability of EBF.ConclusionAmong our 394 isolates, <10% are able to quickly and persistently produce high biofilm levels over 5 h. These isolates belong to a few clones previously described in various studies as dominant gut colonizers in mammalians and birds and comprised the B2-CH40-22-ST131 clone, i.e., the ancestor of the globally disseminated B2-CH40-30-ST131 clone that is the dominant clone among the weak biofilm producers

High Prevalence of ST131 Subclades C2-H30Rx and C1-M27 Among Extended-Spectrum beta-Lactamase-Producing Escherichia coli Causing Human Extraintestinal Infections in Patients From Two Hospitals of Spain and France During 2015

The present study was carried out to evaluate the prevalence of sequence type 131 (ST131) among 188 extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) collected in 2015 in Lucus Augusti University hospital (Lugo, Spain) and AP-HP Beaujon hospital (Clichy, France) with regard to other STs and to characterize, the types of ESBL produced, serotypes, virulence factor (VF)-encoding genes and the ST131 clades and subclades. ST131 was detected in 33 (39.1%) and 46 (47.9%) of the isolates in Lucus Augusti and Beaujon, respectively. The 109 remaining isolates displayed 57 other STs, the following STs being displayed by at least three isolates: ST10 (8 isolates), ST23 (3), ST38 (4), ST58 (3), ST88 (5), ST95 (4), ST167 (3), ST354 (5), ST361 (3), ST410 (6), ST648 (4), ST744 (3), and ST1615 (6). ST354, ST410, and ST1615 were significantly (P < 0.05) more frequent in Lucus Augusti (5.4%, 6.5%, and 6.5%) than in Beaujon (0% for the three STs). The new globally emerging clone ST1193 among extraintestinal clinical ESBL-EC was identified in one isolate from France and one from Spain. CTX-M-15 was the commonest ESBL detected in the two hospitals (44.6% in Lucus Augusti and 50.0% in Beaujon). CTX-M-14 was significantly (P = 0.0003) more frequent in Lucus Augusti (31.5%) than in Beaujon (10.4%), whereas CTX-M-1 (20.8 vs. 7.6%; P = 0.008) and CTX-M-27 (15.6 vs. 6.5%; P = 0.0389) were more frequent in Beaujon than in Lucus Augusti. The ST131 isolates showed a higher virulence score (mean 13.367) compared with the non-ST131 isolates (mean 7.661) (P < 0.001). Among the 79 ST131 isolates, most of them (52; 65.8%) belonged to subclade C2 (also known as subclone H30Rx) followed by those belonging to subclade C1 (cluster C1-M27: 16 isolates, 20.3%; cluster non-C1-M27: 6 isolates, 7.6%) and clade A (4 isolates; 5.1%). The C2 subclade isolates showed a higher VF-encoding gene score (mean 14.250) compared with the C1-M27 cluster isolates (mean 10.875) (P < 0.001). In conclusion, this study highlights the epidemiological differences between the ESBL-EC isolated from two hospitals of France and Spain obtain in 2015 and reports, for the first time, the presence of clone ST1193 in Spain

Clonal Structure, Virulence Factor-encoding Genes and Antibiotic Resistance of Escherichia coli, Causing Urinary Tract Infections and Other Extraintestinal Infections in Humans in Spain and France during 2016

Escherichia coli is the main pathogen responsible for extraintestinal infections. A total of 196 clinical E. coli consecutively isolated during 2016 in Spain (100 from Lucus Augusti hospital in Lugo) and France (96 from Beaujon hospital in Clichy) were characterized. Phylogroups, clonotypes, sequence types (STs), O:H serotypes, virulence factor (VF)-encoding genes and antibiotic resistance were determined. Approximately 10% of the infections were caused by ST131 isolates in both hospitals and approximately 60% of these infections were caused by isolates belonging to only 10 STs (ST10, ST12, ST58, ST69, ST73, ST88, ST95, ST127, ST131, ST141). ST88 isolates were frequent, especially in Spain, while ST141 isolates significantly predominated in France. The 23 ST131 isolates displayed four clonotypes: CH40-30, CH40-41, CH40-22 and CH40-298. Only 13 (6.6%) isolates were carriers of extended-spectrum beta-lactamase (ESBL) enzymes. However, 37.2% of the isolates were multidrug-resistant (MDR). Approximately 40% of the MDR isolates belonged to only four of the dominant clones (B2-CH40-30-ST131, B2-CH40-41-ST131, C-CH4-39-ST88 and D-CH35-27-ST69). Among the remaining MDR isolates, two isolates belonged to B2-CH14-64-ST1193, i.e., the new global emergent MDR clone. Moreover, a hybrid extraintestinal pathogenic E.coli (ExPEC)/enteroaggregative isolate belonging to the A-CH11-54-ST10 clone was identifiedThis study was supported by projects: PI16/01477 from Plan Estatal de I+D+I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad (Gobierno de España) and Fondo Europeo de Desarrollo Regional (FEDER); and ED431C2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, (Xunta de Galicia) and FEDERS

Étude comparative du clade émergent de Escherichia coli ST131 O25b H4 de son clade progéniteur : fitness in vitro et in vivo et formation de biofilm

The clade C of Escherichia coli ST131, an extra-intestinal pathogen (ExPEC) multidrug-resistant, emerged worldwide in the early 2000s. Understanding its expansion is one of the major public health challenges. To contribute to this understanding, we took into consideration the phylogenesis of ST131 and focused our research on comparing the clade C with its progenitor, the clade B, which is composed of strains globally sensitive to antibiotics.The phylogenesis of the clone ST131 describes the diversification of the ancestral clade B into different B subclades (from B1 to B5), B5 giving rise to clade C, which itself has diversified into two subclades, C1 and C2. We wanted to learn about the evolution of these different subclades in terms of relative frequency within all ExPECs. For this purpose, we analyzed the ST131 genomes identified within bacteriemic E. coli systematically collected in England between 2001 and 2012. This analysis showed that, during the studied period, (i) ST131 was one of the few dominant clones, with a dominance of clade C strains, particularly those of subclade C2 and (ii) clade B strains persisted in a stable manner, particularly those of subclades B4 and B5, despite an overall relative frequency lower than that of clade C. Besides, we have compiled a collection of 39 ST131 strains that have been found to be representative of the diversity of B and C clades and subclades, with the exception of one B4 strain (called Hybrid), which carries the fimH30 allele, normally specific to the clade C. Through this collection, we have explored the growth and formation of early biofilm (after 2, 3 and 5 hours of incubation) of clade B and C strains. All strains had equal growth capacities, while they differed in biofilm formation: biofilm was more frequently observed in 2 h in clade B strains than in clade C strains. Then, two representative strains of clades B and C, called Ancestor and Emergent, respectively, as well as Hybrid, were subjected to competitions two by two in vitro and in vivo (in various mouse models). Despite the absence of in vitro fitness differences between these three strains, Emergent was found to be less effective in colonizing the intestinal and/or urinary tract in mice and less virulent in the sepsis model than Ancestor and Hybrid. Referring to the non-functional fimB gene in all strains of clade C, a gene encoding one of the regulators of type 1 fimbriae synthesis involved in biofilm formation and bacterial adhesion, we have deleted it in Ancestor and Hybrid. Although the deletion of the fimB gene abolished in vitro the formation of early biofilm observed in parental strains, no effect was observed when mutants were put in competition with their parental strains, in vitro and in vivo; mutant and parental strain also behaved equally with regard to intestinal colonization and virulence in mice. In total, this work suggests that a global loss of virulence, a process known to improve the level of bacterial transmission, has occurred in ST131 clade C in addition to its acquisition of a multidrug resistance, two evolutions likely to ensure better fitness, especially in environments under antibiotic pressure.Le clade C de Escherichia coli ST131, pathogène extra-intestinal (ExPEC) multirésistant aux antibiotiques, a émergé dans le monde entier au début des années 2000. La compréhension de son essor fait partie des enjeux majeurs de santé publique. Pour participer à cette compréhension, nous avons pris en considération l’histoire phylogénique de ST131 et axé nos recherches sur la comparaison du clade C avec son progéniteur, le clade B, qui est lui composé de souches globalement sensibles aux antibiotiques. L’histoire phylogénétique du clone ST131 décrit la diversification du clade B ancestral en différents sous-clades B (de B1 à B5), B5 donnant naissance au clade C, qui lui-même s’est diversifié en deux sous-clades, C1 et C2. Nous avons souhaité connaitre l’évolution de ces différents sous-clades en termes de fréquence relative au sein de tous les ExPEC. Pour cela, nous avons analysé les génomes de ST131 identifiés au sein de E. coli bactériémiques systématiquement collectés en Angleterre entre 2001 et 2012. Cette analyse a montré que, durant la période étudiée, (i) ST131 faisait partie des quelques clones dominants, avec en son sein une dominance des souches de clade C, en particulier celles de sous-clade C2 et (ii) les souches de clade B persistaient de manière stable, en particulier celles de sous-clades B4 et B5, malgré une fréquence relative globale plus faible que celle du clade C. Par ailleurs, nous avons constitué une collection de 39 souches ST131 qui se sont avérées représentatives de la diversité des clades et sous-clades B et C, à l’exception d’une souche B4 (nommée Hybride), porteuse de l’allèle fimH30, normalement spécifique du clade C. Grâce à cette collection, nous avons exploré la croissance et la formation de biofilm précoce (après 2, 3 et 5 h d’incubation) des souches de clade B et C. Toutes les souches possédaient des capacités de croissance égales, alors qu’elles différaient quant à la formation de biofilm : biofilm plus fréquemment observé en 2 h chez le clade B que chez le clade C. Puis, deux souches représentatives du clade B et du clade C, nommées Ancêtre et Émergente, respectivement, ainsi que Hybride ont été soumises à des compétitions deux à deux in vitro et in vivo (dans divers modèles murins). En dépit de l’absence de différences de fitness in vitro entre ces trois souches, Émergente s’est montrée chez la souris moins bonne colonisatrice des tractus intestinaux et/ou urinaires et moins virulente dans le modèle de septicémie que Ancêtre et Hybride. Faisant référence au gène fimB non fonctionnel chez toutes les souches de clade C, gène codant un des régulateurs de la synthèse des fimbriae de type 1 qui participent à la formation du biofilm et à l’adhésion bactérienne, nous l’avons délété chez Ancêtre et Hybride. Bien que la délétion du gène fimB abolissait in vitro la formation du biofilm précoce observée chez les souches parentales, aucun effet n’a été observé lors de la mise en compétition des mutants avec leurs souches parentales, in vitro comme in vivo ; mutant et souche parentale se comportaient de manière équivalente au regard de la colonisation intestinale et de la virulence chez la souris.Au total, ces travaux suggèrent qu’une perte de virulence globale, processus connu pour améliorer le niveau de transmission bactérien, est survenue chez le clade C de ST131 en plus de son acquisition d’une multirésistance aux antibiotiques, deux évolutions susceptibles de lui assurer un meilleur fitness, notamment dans les environnements sous pression antibiotique

Comparative study of the emerging Escherichia coli ST131 O25b H4 clade and its progenitor clade : in vitro and in vivo fitness and biofilm formation

Le clade C de Escherichia coli ST131, pathogène extra-intestinal (ExPEC) multirésistant aux antibiotiques, a émergé dans le monde entier au début des années 2000. La compréhension de son essor fait partie des enjeux majeurs de santé publique. Pour participer à cette compréhension, nous avons pris en considération l’histoire phylogénique de ST131 et axé nos recherches sur la comparaison du clade C avec son progéniteur, le clade B, qui est lui composé de souches globalement sensibles aux antibiotiques. L’histoire phylogénétique du clone ST131 décrit la diversification du clade B ancestral en différents sous-clades B (de B1 à B5), B5 donnant naissance au clade C, qui lui-même s’est diversifié en deux sous-clades, C1 et C2. Nous avons souhaité connaitre l’évolution de ces différents sous-clades en termes de fréquence relative au sein de tous les ExPEC. Pour cela, nous avons analysé les génomes de ST131 identifiés au sein de E. coli bactériémiques systématiquement collectés en Angleterre entre 2001 et 2012. Cette analyse a montré que, durant la période étudiée, (i) ST131 faisait partie des quelques clones dominants, avec en son sein une dominance des souches de clade C, en particulier celles de sous-clade C2 et (ii) les souches de clade B persistaient de manière stable, en particulier celles de sous-clades B4 et B5, malgré une fréquence relative globale plus faible que celle du clade C. Par ailleurs, nous avons constitué une collection de 39 souches ST131 qui se sont avérées représentatives de la diversité des clades et sous-clades B et C, à l’exception d’une souche B4 (nommée Hybride), porteuse de l’allèle fimH30, normalement spécifique du clade C. Grâce à cette collection, nous avons exploré la croissance et la formation de biofilm précoce (après 2, 3 et 5 h d’incubation) des souches de clade B et C. Toutes les souches possédaient des capacités de croissance égales, alors qu’elles différaient quant à la formation de biofilm : biofilm plus fréquemment observé en 2 h chez le clade B que chez le clade C. Puis, deux souches représentatives du clade B et du clade C, nommées Ancêtre et Émergente, respectivement, ainsi que Hybride ont été soumises à des compétitions deux à deux in vitro et in vivo (dans divers modèles murins). En dépit de l’absence de différences de fitness in vitro entre ces trois souches, Émergente s’est montrée chez la souris moins bonne colonisatrice des tractus intestinaux et/ou urinaires et moins virulente dans le modèle de septicémie que Ancêtre et Hybride. Faisant référence au gène fimB non fonctionnel chez toutes les souches de clade C, gène codant un des régulateurs de la synthèse des fimbriae de type 1 qui participent à la formation du biofilm et à l’adhésion bactérienne, nous l’avons délété chez Ancêtre et Hybride. Bien que la délétion du gène fimB abolissait in vitro la formation du biofilm précoce observée chez les souches parentales, aucun effet n’a été observé lors de la mise en compétition des mutants avec leurs souches parentales, in vitro comme in vivo ; mutant et souche parentale se comportaient de manière équivalente au regard de la colonisation intestinale et de la virulence chez la souris.Au total, ces travaux suggèrent qu’une perte de virulence globale, processus connu pour améliorer le niveau de transmission bactérien, est survenue chez le clade C de ST131 en plus de son acquisition d’une multirésistance aux antibiotiques, deux évolutions susceptibles de lui assurer un meilleur fitness, notamment dans les environnements sous pression antibiotique.The clade C of Escherichia coli ST131, an extra-intestinal pathogen (ExPEC) multidrug-resistant, emerged worldwide in the early 2000s. Understanding its expansion is one of the major public health challenges. To contribute to this understanding, we took into consideration the phylogenesis of ST131 and focused our research on comparing the clade C with its progenitor, the clade B, which is composed of strains globally sensitive to antibiotics.The phylogenesis of the clone ST131 describes the diversification of the ancestral clade B into different B subclades (from B1 to B5), B5 giving rise to clade C, which itself has diversified into two subclades, C1 and C2. We wanted to learn about the evolution of these different subclades in terms of relative frequency within all ExPECs. For this purpose, we analyzed the ST131 genomes identified within bacteriemic E. coli systematically collected in England between 2001 and 2012. This analysis showed that, during the studied period, (i) ST131 was one of the few dominant clones, with a dominance of clade C strains, particularly those of subclade C2 and (ii) clade B strains persisted in a stable manner, particularly those of subclades B4 and B5, despite an overall relative frequency lower than that of clade C. Besides, we have compiled a collection of 39 ST131 strains that have been found to be representative of the diversity of B and C clades and subclades, with the exception of one B4 strain (called Hybrid), which carries the fimH30 allele, normally specific to the clade C. Through this collection, we have explored the growth and formation of early biofilm (after 2, 3 and 5 hours of incubation) of clade B and C strains. All strains had equal growth capacities, while they differed in biofilm formation: biofilm was more frequently observed in 2 h in clade B strains than in clade C strains. Then, two representative strains of clades B and C, called Ancestor and Emergent, respectively, as well as Hybrid, were subjected to competitions two by two in vitro and in vivo (in various mouse models). Despite the absence of in vitro fitness differences between these three strains, Emergent was found to be less effective in colonizing the intestinal and/or urinary tract in mice and less virulent in the sepsis model than Ancestor and Hybrid. Referring to the non-functional fimB gene in all strains of clade C, a gene encoding one of the regulators of type 1 fimbriae synthesis involved in biofilm formation and bacterial adhesion, we have deleted it in Ancestor and Hybrid. Although the deletion of the fimB gene abolished in vitro the formation of early biofilm observed in parental strains, no effect was observed when mutants were put in competition with their parental strains, in vitro and in vivo; mutant and parental strain also behaved equally with regard to intestinal colonization and virulence in mice. In total, this work suggests that a global loss of virulence, a process known to improve the level of bacterial transmission, has occurred in ST131 clade C in addition to its acquisition of a multidrug resistance, two evolutions likely to ensure better fitness, especially in environments under antibiotic pressure

Diversity and functionality of plasmid-borne VagCD toxin–antitoxin systems of Klebsiella pneumoniae

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Laboratory-based strategy using a new marketed polymerase chain reaction assay to manage diarrheic episodes among patients from rehabilitation and long-term care facilities

Management of Norovirus and Clostridium difficile gastroenteritis is challenging for rehabilitation and long-term care facilities. We evaluated the contribution of a 2-step laboratory-based strategy, including a new ready-to-use Norovirus polymerase chain reaction assay to promote isolation precautions. C difficile and Norovirus were successively identified from 17% and 23% of 52 episodes of diarrhea, respectively, during the winter season, leading to 100% adequate isolation measures. In patient populations with numerous risk factors for diarrhea, a combined laboratory-based approach could improve infection control

Emergence of the C1-M27 cluster in ST131 Escherichia coli from companion animals in France

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In-vivo loss of carbapenem resistance by extensively drug-resistant Klebsiella pneumoniae during treatment via porin expression modification

International audienceKlebsiella pneumoniae, an Enterobacteriaceae that mostly causes hospital-acquired infections, belongs to the recently published WHO's list of antibiotic-resistant pathogens that pose the greatest threat to human health. Indeed, K. pneumoniae is the enterobacterial species most concerned by both resistance to extended-spectrum cephalosporins, due to extended-spectrum β-lactamase (ESBL) production, and resistance to carbapenems, i.e. the β-lactams with the broadest activity. Carbapenem resistance is related not only to carbapenemase production, but also the production of ESBL or AmpC and the loss of general porins. Here, we characterized the mechanisms that deprived a urinary ESBL-producing, porin-deficient K. pneumoniae isolate, isolated 13 days after the end of a 40-day course of imipenem treatment, of its carbapenem resistance. These mechanisms were observed in two in-vivo derivatives of this isolate and consisted of mutations in genes encoding molecules that participate in the downregulation of the synthesis of PhoE, a porin specialized in phosphate transport. We obtained three new derivatives from one of the two original derivatives, following in-vitro antibiotic pressure, in which the carbapenem resistance was restored because of mutations in genes encoding molecules that participate in the upregulation of PhoE synthesis. Thus, we uncovered novel mechanisms of carbapenem resistance/susceptibility switching in K. pneumoniae

Success of Escherichia coli O25b:H4 Sequence Type 131 Clade C Associated with a Decrease in Virulence

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